Analyses of molecular fractions of biologically significant molecules in desirable forms from biological origins have always been an important task for biochemical research. Molecules, such as ribonucleic acid (RNA), deoxyribonucleic acid (DNA) or cDNA, protein, peptide, lipids or polysaccharides, are usually required to be fractionated based on their biochemical properties such as size and charge before further accurate or meaningful analyses. Fractionating methods are those that can fractionate molecules and generate a spectrum or a serial fractions of molecules based on one or more properties of molecules, which methods include agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), two-dimensions PAGE, isoelectrical focusing, ion exchange chromatography, filtration chromatography, hydrophobic chromatography, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), gas chromatography, atomic absorption, affinity chromatography or gradient centrifugation. With above fractioning methods, molecules can be fractionated according to their properties, such as molecular size, charges, organic phase solubility, affinity, mass, shape, density or color.
For examples, the conventional Northern blot analysis and Western analysis are the most widely used and vital techniques for analyzing sizes and amounts of RNA or protein molecules from biological origins. Northern blot (for RNA analysis) and Western blot (for protein analysis) are named after Southern blot (for DNA analysis), (Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517, which is incorporated herein by reference). They have been the must or ultimate assays of choice to detect the sizes and expression level of certain genes in some cell populations or tissues. Although there are some other methods, such as differential display, RT-PCR or RNA protection assay (RPA) that can be used for part of this purpose, Northern blot analysis and Western blot analysis are the distinguished assays that can determine the molecular size or weight of the target gene or protein. The other methods are generally less favorable and more disadvantageous as they tend to produce unreliable results introduced as a result of complicated or enzymatic manipulation. Since Northern blot analysis and Western blot analysis are the closest methods to confirm real-life situation of gene or protein expression because they are rather direct measurement than other methods, they are ultimately important tools in determining sizes of alternative spliced gene, post translational modification of proteins as well as levels of gene or protein expression.
It has been many years for people to make and use conventional Northern blot and Western blot. Up to today, the ways to make conventional Northern blot analysis and Western blot are still the same as many years ago. When making Northern blot, RNA electrophoresis is performed using agarose gel with denaturing agents such as formaldehyde and formamide. Since RNA molecules carry one negative charge per molecule, their migration in agarose gel depends on the length of nucleic acids or the size of the molecules. Fractionated nucleic acids on the gel can be blotted to nylon or nitrocellulose membranes to make Northern blot. Probes that contain labeled nucleotides and are complementary to the target nucleic acid chain can be added for hybridization and detection of specific RNA fraction in Northern blot. It is Northern blot analysis to make it possible for estimating the size and the relative amount of a fraction of RNA simultaneously.
It is different to make Western blot from Northern blot because properties of protein are different from RNA. Proteins may carry negative or positive charges, depending on their amino acid compositions. In order to generate a uniform spectrum or serial fractions of proteins based on the molecular weight, sodium dodecyl sulphate (SDS), dithiothreitol (DTT) and β-mecarptoethanol are added to protein loading buffer and the gel. SDS is an anionic detergent that binds to proteins fairly specifically in a mass ratio of 1.4:1 and confers a negative charge to the protein in proportion to its length. All protein molecules become negative charged uniformly in present of SDS. In addition, reducing reagents such as dithiothreitol (DTT) and β-mecarptoethanol can disrupt the disulfide bonds of protein molecules to overcome the affect on gel electrophoresis by non-uniformed structures of protein. After gel electrophoresis, the gel can be stained or the protein can be transferred to a membrane to make Western blot. To detect particular fractions of proteins on Western blot, antibodies specific for the target proteins (antigens) are incubated with the membrane. The bound antigen-antibody complex is further detected by chemiluminescent, fluorescence or radioactive method. As Northern blot analysis, Western blot analysis is also capable of determining the size and relative amount of target proteins simultaneously.
While the conventional Northern blot analysis and Western blot analysis are the major tools for the afore-mentioned purposes, they are nevertheless limited as to the number of samples that can be applied on one membrane. It is generally agreed that fewer than ten samples can be accommodated on one membrane, which limitation renders the use of Northern or Western blot analysis for a large-scale, high throughput analysis of molecular fractions unfeasible.
The demands for high throughput analysis of molecular fractions become urgent with the discovery of more than 30,000 new genes due to the completion of the human genome project. U.S. Pat. Nos. 6,582,969 and 6,576,478 (Wagner et al) disclose a method and microdevices for high throughput screening of biomolecules. These microdevices comprise a certain arrayed of specially micromachined or microfabricated channels with a plurality of built-in noncontiguous reactive sites that are provided with biological moieties immobilized on a monolayer via an affinity tag linked to a substrate. These microdevices are capable of screening functionality or structurally related components passing through the channels. U.S. Pat. No. 6,537,749 (Kuimelis et al) discloses addressable protein arrays in which arrays of nucleic acid-protein fusions are immobilized to a solid surface through a linker layer that is provided with a spacer group on one end and an oligonucleotide sequences on the other end for interacting with an addressable array of molecules complementary to the immobilized oligonucleotide sequences.
While these arrayed techniques may be sufficient to provide high throughput screening of biomolecules, they have to be specially constructed with desirable immobilizing agents and affinity groups. In addition, none of these techniques aims at the incorporation of the advantages of conventional Northern blot analysis and Western blot analysis as described above, such as size of mRNA or protein molecules, while improving their shortcomings to develop a high throughput screening system capable of screening a substantial amount of samples with more detection sensitive and consuming less treasured materials.
For the foregoing reasons, there is an apparent need for a method and device for high throughput screening of molecular fractions that combines the unique features of molecular fractionating technique and the power of micro-array.
There is a further need for a method and device for high throughput screening of molecular fractions that creates a more sensitive method in detection of certain desirable biological activity while consuming less treasured materials
Accordingly, this present invention provides a solution to create a high throughput screening system, such as incorporating both Northern blot analysis and Western blot analysis as illustrated above, while provided with molecular fractionating methods and properties based to fractionate molecules fundamentally different.